1. Local environment- sensitive two-photon dye Nikolay S. Makarov, Erich Beuerman, Mikhail Drobizhev, Aleksander Rebane Department of Physics Montana State University, Bozeman, MT Jean Starkey Department of Microbiology Montana State University, Bozeman, MT

2. We present a detailed study of the local environmental sensitivity of the commercially available laser dye, Styryl-9M. Positions of the one-photon and two-photon absorption maxima and two-photon absorption minimum of the dye are sensitive to the solvent polarity. In aqueous solution its absorption and fluorescence spectra consist of two peaks whose relative strength depend on the surrounding pH-s. The dye shows one of the highest two-photon absorption cross sections, 700- 1300 GM at the peak, among widely available compounds. Comparison of the linear and nonlinear properties shows that its maximum cross section can be described by an effective two-level model. Based on the properties of Styryl-9M we propose a new method of sensing local environment polarity in solutions and biological phantoms. We show that the dye is a promising candidate for two-photon biological imaging and microscopy. KEYWORDS: TWO-PHOTON ABSORPTION, STYRYL-9M, PH SENSOR, POLARITY SENSOR, BIOLOGICAL IMAGING Abstract

3. Outline Advantages of two-photon microscopy Properties of molecular probes for two-photon microscopy Two-photon absorption of Styryl-9M Linear photophysical properties of Styryl-9M pH sensitivity of Styryl-9M One-photon sensitivity to local polarity Two-photon sensitivity to local polarity Two-photon sensitivity to local environment in biological phantoms

4. Advantages of two-photon microscopy Inherent 3D resolution Larger penetration depth Lower scattering Lower photodamage Lower absorption by intrinsic molecules Lower autofluorescence background Probes for two-photon microscopy Endogenous fluorophores (NADH, NADPH, retinol, lipofucsin, etc.) + natural source of 2PA- excited fluorescence + fills the whole visible spectrum - low 2PA cross sections Fluorescent proteins + can be genetically encoded + high flexibility studying small animals - small loading concentration - substantial size - faster photobleaching - higher cost Exogenous fluorophores + high 2PA cross sections + possible targeting + high stability + small size + possible near-infrared fluorescence - delivery problems - non-specific dying

5. Two-photon absorption, chloroform

6. Polarity dependence of 2PA

7. Perrin plot for Styryl-9M

8. Solvatochromic Stokes shifts for Styryl-9M

9. Fluorescence decay kinetics for Styryl-9M  Methanol =410 ps  Ethanol =570 ps  Ethylene glycol =470 ps

10. Two-level model description of the two-photon cross section in chloroform

11. pH sensitivity of Styryl-9M: absorption

12. pH sensitivity of Styryl-9M: fluorescence

13. pH sensitivity of Styryl-9M

14. Imaging layout Direction of the laser beam

15. One-photon sensitivity to local polarity 1 – 2-chlorobutane; 2 – dichloromethane; 3 – pentanal; 4 – isopropanol; 5 – ethylene glycol; 6 – acetone; 7 – ethanol; 8 – mixture of 70% ethanol + 30% DI water; 9 – mixture of 50% ethanol + 50% DI water; 10 – acetonitrile; 11 – mixture of 30% ethanol + 70% DI water Two “unknown” samples: (1) mixture of 50% 2-chlorobutane + 50% isopropanol (2) mixture of 40% ethanol + 60% DI water The determined polarity function is 0.386 for (1) and 0.497 for (2) which is less than 2% off the “true” values

16. Two-photon sensitivity to local polarity

17. 2PA sensitivity to local environment in biological phantoms 1 – 10  l of 5mg/ml Styryl-9M dissolved in DMSO, 5  10 5 Mouse embryo fibroblast cells, 0.6 ml setting solution, 3 ml rat tail collagen, 1  l linoleic/oleic acid mixture, 1 ml serum and phenol red –free medium overlay 2 – 25  l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen 3 – 25  l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen, 1  l linoleic/oleic acid mixture 4 – 25  l of 1mg/ml Styryl-9M dissolved in DMSO, 0.8 ml setting solution, 4 ml rat tail collagen, 1  l liposin

18. Conclusions Styryl-9M shows one of the strongest two-photon cross section among commercially available chromophores Despite the lowest energy absorption peak consists of several broadened transitions, it can be considered as an effective two- level system Both absorption and fluorescence spectra of the dye are highly sensitive to pH Both 1PA and 2PA peaks of the dye are sensitive to the solvent polarity These properties can be used for two-photon sensing of local environment in biological systems

19. Self-advertisement Graduating in about 1 year. Looking for a postdoctoral position in fields of nonlinear optics of organic molecules, two-photon biological imaging and microscopy, biophotonics, two-photon photolithography, and photodynamic therapy. Soros student 2000, 2001, 2002, 2003 Russian Federation’s President grant 2001 Diploma of Ministry of Education RF for the best scientific student work in natural, technical and humanitarian sciences 2001 RFBR travel grants 2002 Grant of Saint-Petersburg administration for students, aspirants and young specialists-2002 Medal of Russian Academy of Science for the best student work in general physics and astronomy 2002 SPIE Scholarship grant 2003 Diploma for best university graduating student 2003 Dynasty foundation grant 2003 Medal of Ministry of Education RF for the best scientific student work in natural, technical and humanitarian sciences 2003 Grant of Saint-Petersburg administration for students, aspirants and young specialists 2004 Soros aspirant 2004 SPIE Scholarship grant 2006 SPIE BACUS Photomask Scholarship 2007 SPIE D.J. Lovell Scholarship 2008 Grants and awards Important publications Makarov N.S., Drobizhev M, Rebane A, “Two-photon absorption standards in the 550-1600 nm excitation wavelength range”, Opt. Expr., 16, 2008, 4029-4047. Makarov N.S., Rebane A., Drobizhev M., Wolleb H., Spahni H., “Optimizing two-photon absorption for volumetric optical data storage”, J. Opt. Soc. Am. B, 24, 2007, 1874-1885. Makarov N.S., Bespalov V.G., “Effective method of anti-Stokes generation by quasi-phase-matched stimulated Raman scattering”, J. Opt. Soc. Am. B, 22, 2005, 835-843. Drobizhev M., Makarov N.S., Hughes T., Rebane A., “Resonance Enhancement of Two-Photon Absorption in Fluorescent Proteins”, J. Phys. Chem., 111, 2007, 14051-14054. Rebane A., Makarov N.S., Drobizhev M., Spangler B., Tarter E.S., Reeves B.D., Spangler C.W., Meng F., Suo Z., “Quantitative prediction of two-photon absorption cross section based on linear spectroscopic properties”, J. Phys. Chem. C, 112, 2008, 7997- 8004.